Cellular metabolic energy is stored in the form of neutral lipids, particularly triacylglycerols (TGs), which are packaged in cytoplasmic lipid droplets (LDs). LDs also contain sterol esters (SEs), which are required for membrane biogenesis. Excessive accumulation of LDs occurs during the progression of certain diseases, including obesity, metabolic syndrome and atherosclerosis. LDs are also required for hepatitis C virus replication, and a number of viral proteins specifically interact with this organelle. Despite their significance to human health, surprisingly little is known about basic LD cell biology, especially in terms of protein targeting. The hydrophobic core of LDs consisting of TGs and SEs is bounded by a phospholipid monolayer, which harbors a set of largely-unidentified proteins. Most functions of LDs, including TG synthesis, TG storage, and energy mobilization, are executed and regulated by these surface proteins. Among organelles, LDs are unique because their surface is an apposition of a hydrophobic phase (the LD core) and an aqueous phase (the cytoplasm). This monolayer is thus not configured to accommodate typical transmembrane proteins with globular domains flanking transmembrane segments. Therefore, the targeting of particular proteins to LDs must involve unique mechanisms, which are the subject of the research proposed here. To elucidate these mechanisms, we must first determine a high-confidence LD proteome, which we can accomplish via our state-of-the-art quantitative mass spectrometry-based proteomics methods. We will then proceed to determine how proteins are targeted to LDs from the cytoplasm, focusing on a set of model proteins and applying a variety of cell biological and biochemical methods we have established in our laboratory. We will subsequently use our unbiased proteomics approach to define the scope of proteins that share the pathways defined for these models. In a second line of research, we propose to determine how a distinct set of proteins that contain sequences predicting multiple trans-membrane domains are targeted to LDs or closely- associated membranes. For each part of the project, we will test the functional consequences of LD protein targeting as well as evolutionary conservation of the mechanisms we discover. Although the research proposed here is basic, our determination of the fundamental cellular mechanism that drives lipid droplet protein targeting will facilitate the development of therapeutic strategies to combat diseases that involve lipid droplets, as well as propel further research into the cell biology of these organelles.